The use of monoclonal antibody (mAb) molecules that comprise Fc peptide chains as therapeutic reagents has become an effective approach for the treatment of various diseases. Antibody-derived molecules that contain Fc peptide chains, such as mimetibody molecules, described infra, and Fc fusion molecules, also have therapeutic potential. In addition, these molecules are useful research tools for gaining a better understanding of the immunopathogenesis of various diseases as well as other biological processes.
Large-scale production and purification of antibody, mimetibody, and Fc fusion molecules is necessary to prepare these Fc peptide chain molecules for therapeutic or research use. A commonly encountered problem in the production and purification of such molecules is the formation of soluble high molecular weight aggregates comprising multiple Fc peptide chain molecules (e.g., aggregates of more than two unaggregated, low molecular weight antibody or mimetibody molecules). The presence of such aggregates is highly undesirable in therapeutic preparations, because of the risk that some patients will suffer life-threatening immune reactions in response to receiving aggregate. Additionally, Fc peptide chain aggregates can cause the formation of undesirable insoluble precipitates in Fc peptide chain preparations intended for research or therapeutic uses.
One goal during the purification of Fc peptide chain molecules is to obtain an unaggregated Fc peptide chain product (e.g. unaggregated, low molecular weight antibody or mimetibody molecules) in high yield that is essentially free of aggregated molecules. Typically, a preparation of purified Fc peptide chains containing less than 5% aggregate at a total percent yield of Fc peptide chains greater than 70% is desired.
One of the first steps in the production and purification of Fc peptide chain molecules is normally a protein A chromatography resin based purification step. Protein A chromatography resins comprise protein A peptide chains that specifically bind Fc peptide chains. This binding activity does not discriminate between aggregated or unaggregated Fc peptide chains—both are bound to protein A chromatography resins. Importantly, protein A chromatography resins separate Fc peptide chains from other molecules on the basis of this specific binding. Such resins do not separate Fc peptide chains from other molecules or complexes on the basis of molecular weight differences. Consequently, in conventional protein A chromatography methods both aggregated, high molecular weight Fc peptide chains and unaggregated, low molecular weight Fc peptide chains are released from the protein A chromatography resin during an elution step. This produces an aqueous solution comprising large quantities of both undesirable, aggregated Fc peptide chain molecules and desirable, unaggregated Fc peptide chain molecules.
One consequence of the inability of conventional protein A chromatography methods to separate aggregated and unaggregated Fc peptide chain molecules is that several additional purification processes such as size exclusion chromatography or ion exchange chromatography techniques are needed to obtain preparations of unaggregated Fc peptide containing less than 5% aggregate. This subsequent processing is expensive, time consuming, and causes total percent yield values to decrease well below 70%.
Thus, a need exists for protein A chromatography-based purification methods that produce purified Fc peptide chain preparations containing less than 5% aggregate at total percent yield values greater than 70% without the use of additional purification processes.